Microbiological oxidation of substituted naphthalenes



United States Patent 3,340,155 MICROBIOLOGICAL OXIDATION OF SUBSTITUTEDNAPHTHALENES John D. Donros, J12, Fanwood, N.J., and Richard L.

Raymond, Wilmington, Del. assignors to Sun Oil Company Philadelphia,Pa., a corporation of New Jersey No Drawing. Filed Sept. 18, 1963, Ser.No. 309,889 7 Claims. (Cl. 195-28) This invention relates to themicrobiological oxidation of alkyl napthalenes to form the correspondingmonoacids. More particularly, this invention relates to novel processesfor preparing alkyl naphthalene monocarboxy acid in high yield by themicrobiological oxidation of one of the alkyl groups of a dialkylnaphthalene substrate.

While the use of microbiological means in the conversion of complexorganic compounds is well-known, such methods have rarely beensuccessful in converting hydrocarbons to useful compounds. Most often,microbiological oxidation of hydrocarbons has resulted in the conversionof these hydrocarbons to carbon dioxide and 'water, so that theeffective microbiological oxidation of any hydrocarbon is at best ahighly unpredictable art. Amongst those hydrocarbons whose oxidationproducts are of considerable commercial value are alkyl naphthalenes,the carboxylic acid derivatives of which are useful as antifungalagents. Heretofore, these compounds, as for example the monocarboxylicacid of an alkyl naphthalene such as methyl naphthalene, have beenprepared by the elaborate and expensive chemical oxidation of thecorresponding dimethyl naphthalenes such as, for example, 2,6-dimethylnaphthalcne, and methods have long been sought for convertingsuch compounds by less expensive means and in higher yield.

It has now been found, in accordance with the present process, that theoxidation of dialkyl naphthalenes to form the corresponding monoacids inhigh yield may be achieved microbiologically by subjecting said alkylnaphthalenes to the oxygenating action of a microorganism of the genusStreptomyces, and particularly the species S. achromogenes ATCC 15,077.

The substrates employed in this invention are naphthalenes having twoalkyl groups substituted for each of two different hydrogen atoms on thenucleus. The substituent alkyl groups can be the same or different andmay be branched or straight-chain groups having from 1-6 carbon atoms.Surprisingly, it has been found that when the alkyl side-chain containsan odd number of carbon atoms, the resulting acid moiety will containonly one carbon atom, whereas when the alkyl side-chain contains an evennumber of carbon atoms the corresponding naphthalene acetic acidderivative is formed. Illustrative examples of the dialkyl naphthalenesubstrates which may be used to prepare the products of this inventionare: 1,2-dimethyl-naphthalene; 1,3-diethyl-naphtha1ene;1,4-dipropylnaphthalene; 1,5-dibutyl-naphthalene;2,6-dimethyl-naphthalene; 2,7-dimethyl-naphthalene;1-methyl-8-ethyl-naphthalene; 2-ethyl-3-propyl-naphthalene;2-propyl-6-butylnaphthalene; 2-butyl-7-pentyl-naphthalene and the like,and mixtures thereof.

When the aforementioned starting materials are thus treated inaccordance with this invention, there are obtained the correspondingalkyl-substituted monoacids, as for example, 6-methyl-2-naphthoic acid,7-methyl-2-naphthoic acid, 6-propyl-2-naphthoic acid,7-ethyl-2-naphthylacetic acid, 1-rnethyl-2-naphthoic acid and the like.

In the practice of this invention, the novel microbiological oxidationprocess is desirably carried out under conditions used in conventionalaerobic fermentations known to the art. Thus, for example, a suitablemineral saltshydrocarbon medium is inoculated with a viable strain ofthe microorganism Streptomyces archromogenes, and

aerated and agitated under submerged conditions during the incubationperiod of from about two to eight days and preferably for about five tosix days. The pH of the fermentation medium may vary from about 4.5-8.5,with a preferred range of from about 6.8-7.2. The temperature at whichthe fermentation is carried out may range from 2540 C. and preferably isfrom 26-37 C.

As in conventional submerged aerobic carbohydrate fermentations,aeration, and dispersion of the broth during fermentation is essential.The aeration can be controlled by passing air or oxygen-nitrogenmixtures through the broth in either fine, coarse, or as both fine andcoarse bubbles. The aeration should be coupled with a means of agitatingor dispersing the broth. For example, the broth can be aerated by theabove method while rotating or tumbling the fermentor or *while stirringvigorously with an impeller or propeller type stirrer. In any event, :aconsistent means of dispersing and aerating the broth duringfermentation is necessary.

Where the hydrocarbon substrates are volatile, it is preferred to use aclosed system to insure maximum utili zation of the substrate by themicroorganism and to prevent loss of the alkylated naphthalene substrateto the atmosphere. An adequate sterile air supply should be maintainedduring the oxidation by the submerged cultures. If desired, thesubstrate vapors and gases produced during fermentationcan beventedoif,recovered,'and recycled back to the fermentation medium.

The quantity of hydrocarbon substrate employed in this process may rangefrom 1 to 9% of the total nutrient medium and preferably should be fromabout 3 to 7%. It is desirable in carrying out this process that thedialkyl naphthalene substrate be introduced into the fermentation brothin amounts below growth-limiting concentrations. Thus, for example, atthe beginning of the ferment-ation, as little as 0.2% of the alkylnaphthalene may be added, followed by an additional 1% after the first24 hours, another 1% after 48 hours, and the remainder up to 9% after anadditional .24. hours. Depending upon the total amount of dialkylnaphthalene to be utilized, however, proportionably larger amounts maybe added at 12 to 24 hour intervals until the fermentation is complete.

The resulting monoacids are conveniently recovered from the fermentationbroth by first adjusting the pH of the broth to about -10, preferablywith a strong base such as NaOH, at which pH the monoacid is solublewhile the unconverted alkyl naphthalene is not. Following filtration,the filtrate may then be treated with a strong acid such as HCl until,at a pH of 2.5 or below, the monoacid is precipitated out, recovered byfiltration, and further purified, if necessary, by known methods.

A suitable nutrient medium for the oxidative fermentation should containa source of carbon, nitrogen, and mineral elements. Also, since themicroorganisms used in this transformation are incapable of utilizingthe alkylated naphthalene substrates as their sole source of carbon,carbohydrate sources such as sugars, starches, preferably pearl starch,paraffinic hydrocarbons (straight chain C C and the like must be addedto the medium. Suitable sources of nitrogen include natural productssuch as yeasts, corn, steep, liquor, cotton, seed, meal, beef extract,enzymatically digested proteins, various proteinaceous products such aspeptones and amino acids, as well as ammonium salts, nitrates, nitrites,quaternary bases and salts, urea, and the like. The minerals andelements required for the fermentation such as phosphorus, magnesium,and trace metals can be obtained from the appropriate natural productsused as nitrogen sources or can be added in the necessary amount as thesalts and ions of the metals.

While the organisms described in this application are capable of goodgrowth in a simple mineral salts-hydro- Percent P.T. pearl starch 0.1Urea 0.1

VN32HPO4 0.6 KH PO 0.4 MgSO, 0.08

The inoculum for the fermentation can be prepared in several ways. Forexample, a soil sample in which the organism is found can be sprinkledon a mixture of paraffinic hydrocarbons and minerals until itsprefermentative growth is attained. Alternatively, it can be suspendedin water and grown on a conventional carbohydrate-proteinaceous-mineralmedium, or it can be grown on a medium such as an agar slant. Suchinocula may then be used to inoculate other batches of sterile media infermentor tanks. A preferred medium for preparing the inoculum is asfollows:

Percent Cerelose 0.1 Yeast extract 8-50 0.1 Urea 0.1 NaHPO 0.6 KH PQ,0.4 MgSO, 0.08 2,6-dimethylnaphthalene 0. 1

The inoculum employed in the practice of this invention may consist notonly of the growing cells of the microorganism, but also washedsuspensions of these growing cells, either in their growth phase orstationary phase, or as resting cells.

The following is a description of the morphological and culturalcharacteristics of the previously-undescribed Streptomyces speciesemployed in this process, a culture of which has been deposited in theAmerican Type Culture Collection in Washington, DC, where it wasassigned the above-designated ATCC Number. This culture was isolatedfrom oil-soaked soil in the Marcus Hook Refinery of the Sun Oil Company,Marcus Hook, Pa. it possesses the bacteriological characteristicsdetailed below, and While it is similar to Streptomyces achromogenesdescribed in Bergeys Manual of Determinative Bacteriology, 7th Edition,it cannot be identified as being the same as this organism, and is,therefore, considered to be a new species:

4 (15) Carbohydrate test in phenol red broth using 0.5%

specific carbohydrate substrate (a) Mannit-olNo acid; no gas (b)LevuloseAcid, no gas (c) LactoseNo acid, no gas (d) Sorbitol No acid, nogas (e) SaccharoseNo acid, no gas (f) Arabinose-NO acid, no gas g)Maltese-No acid, no gas Example I.Preparatz'0n of 6-methyl-2-naphthoicacid from 2,6-dimethylnaphthalene A basal nutrient medium prepared froma commercial trace metals solution is combined with the following majorcomponents:

Percent by weight 1.0

Glucose Urea 0.1 MgSO, 0.08 Phosphate buffer 0.1

The medium is brought to 1000 ml. volume with tap water and thensterilized by autoclaving at C. for 45 minutes. It has a pH of 6.8. Theinoculum is prepared by cultivating a viable strain of S. achromogenesATCC No.

15,077 in a commerical beef broth nutrient solution and Percent byweight a 0 1 Urea MgSO, 0.08 Phosphate buffer 0.1 Yeast extract a r 4 aI 0.1 2,6-dimethylnaphthalene 0.1

After bringing up the volume of the broth to 1000 ml. with tap water,the fermentation is allowed to proceed at a pH of 7.2 at 30 C. for 120hours. At the end of this time the pH of the broth is adjusted to 9.0,and the mycelia and unconverted starting material filtered off anddiscarded. After removal of the mycelia the filtered broth is acidifiedto pH 2.5 with HCl and a crystalline product precipitates out. Analysisindicates the precipitate is 6 methyl-Z-naphthoic acid which can befurther purified by a variety of methods well known to the art, as, forexample, the following:

The precipitated product is extracted into diethyl ether, back extractedinto 1 N NaOH solution and decolorized by treatment with activatedcarbon. After removal of the carbon, the aqueous solution is treatedwith dilute HCl until it is neutral or only slightly basic. Then thesolution is chilled to about 5 C. overnight and allowed to warm up withvigorous stirring while adding a small quantity of tertiary butylchloride. The gradual hydrolysis of the butyl chloride to 'HCl producesthe proper degree of acidity to precipitate the product in the form oflarge crystals. These are washed and the traces of butanol removed undervacuum. Infrared analysis, melting point, and partition chromatographyconfirm the products iden= tity as 6-methyl-2-napht-hoic acid.

Example II.-Preparation of 7-methyl-2-naphth0ic acid from2,7-dimethylnaphthalene Streptomyces achromo'genes (ATCC 15,077) isgrown on hexadecane in the same basal mineral medium as is employed inExample I at 30 C. for four days under aerobic conditions. The cells areharvested by centrifugation and washed with M/30 phosphate buffer pH7.0. They are then resuspended in M/30 buffer to give a concentration ofcell material of, 12.4 mg./m1. (dry weight). One ml. of this suspensionis placed. in a Warburg flask with 20 mg. of 2,7-dimethylnaphthalene in1 ml. of M/ 30 phosphate buffer. This mixture is shaken aerobically for21 hours at 303 C. in a water bath. At the conclusion of the experiment,the cells are removed by filtration on a sintered glass filter. Theclear filtrate which has a pH of 6.9 is adjusted to pH 3.0 with HCl toyield a precipitate of 7-methyl-2-naphthoic acid.

Example III.Preparati0n of other alkyl naphthoic acids fromdialkylnaphthalene substrates Using the same procedures and techniquesdescribed in Examples I and II, the following transformations ofdialkylnaphthalene substrates to the corresponding alkylnaphthoic acidproduct are accomplished in good yield. In all cases identity of theproducts is confirmed by melting point, infrared analysis, andchromatography.

Substrate 2,6-dipropylnaphthalene 2,7 -diethylnaphthalene spores andmycelia are transferred from an agar slant to a 25 0 ml. portion of thenutrient broth given below:

Component: Percentage by weight Bacto-soytone 1.0 Bacto-dextrose 4.0

Deionized water to volume.

The 250 m1. portions of fungi and broth are placed in steriletrypsinizing flasks (500 ml.) and the flasks placed on a rotary shakerfor 72 hours at room temperature. At the end of this incubation timeperiod, 20 ml. aliquots of the liquid are homogenized and an aliquotplaced into each one of 4 sterile trypsinizing flasks (300 ml.)containing ml. of the above nutrient broth. To two of the flasks areadded 250 p.p.m., respectively, of the alkylnaphthoic acids of ExamplesI and 11 being evaluated The other 2 untreated flasks are used ascontrols for comparison purposes. The 4 flasks (2 treated and 2untreated) are placed on a rotary shaker operating at 240 rpm. at roomtemperature for 3 days. After the second incubation time the flasks areremoved for visible fungal growth.

RESULTS The two products from Examples I and H give substantiallycomplete inhibition of fungal growth at 250 ppm. evidencing their valueas anti-fungal agents.

The invention claimed is:

1. A process for the production of alkyl naphthalene monocarboxy acidwhich comprises subjecting a dialkyl naphthalene having from 1 to 6carbon atoms in each alkyl group to the oxygenating activity ofStreptomyces achromogenes ATCC 15,077 in an aqueous nutrient mediumunder aerobic conditions and recovering the corresponding alkylnaphthalene monocarboxy acid.

2. A process according to claim 1 wherein each of said alkyl groups has1 to 2 carbon atoms.

3. A process according to claim 2 wherein each of said alkyl groups ismethyl.

4. A process according to claim 3 wherein said dialkyl naphthalene is2,6-dimethylnaphthalene and 6-methyl-2- naphthoic acid is recovered.

5. A process according to claim 3 wherein said dialkyl naphthalene is2,7-dimethylnaphthalene and 7-methy1-2- naphthoic acid is recovered.

6. A process according to claim 2 wherein each of said alkyl groups isethyl.

7. A process according to claim 6 wherein said dialkyl naphthalene is2,7-diethylnaphthalene and 7-ethyl-2-naphthylacetic acid is recovered.

No references cited.

ALVIN E. TANENHOLTZ, Primary Examiner.

1. A PROCESS FOR THE PRODUCTION OF ALKYL NAPHTHALENE MONOCARBOXY ACIDWHICH COMPRISES SUBJECTING A DIALKYL NAPHTHALENE HAVING FROM 1 TO 6CARBON ATOMS IN EACH ALKY GROUP TO THE OXYGENATING ACTIVITY OFSTREPTOMYCES ACHROMOGENES ATCC 15,077 IN AN AQUEOUS NUTRIENT MEDIUMUNDER AEROBIC CONDITIONS AND RECOVERING THE CORRESPONDING ALKYLNAPHTHALENE MONOCARBOXY ACID.